Photothermal imaging device and system

ABSTRACT

Mid-infrared photothermal heterodyne imaging (MIR-PHI) techniques described herein overcome the diffraction limit of traditional MIR imaging and uses visible photodiodes as detectors. MIR-PHI experiments are shown that achieve high sensitivity, sub-diffraction limit spatial resolution, and high acquisition speed. Sensitive, affordable, and widely applicable, photothermal imaging techniques described herein can serve as a useful imaging tool for biological systems and other submicron-scale applications.

CROSS REFERENCE TO RELATED APPLICATION

This application is a non-provisional application claiming priority fromU.S. Provisional Application Ser. No. 62/318,698, filed Apr. 5, 2016,entitled “SUPER-RESOLUTION MID-INFRARED PHOTOTHERMAL IMAGING” andincorporated herein by reference in its entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Grant No. CHE1110560 awarded by the National Science Foundation (NSF), Grant No.N00014-12-1-0130 awarded by the Office of Naval Research (ONR), andGrant No. W911NF-14-1-0604 awarded by the U.S. Army Research Office. Thegovernment has certain rights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to mid-infrared imaging. Morespecifically, the present disclosure relates to imaging structures andobjects with a sub-micron spatial resolution using mid-infrared light.

BACKGROUND OF RELATED ART

Mid-infrared (mid-IR or MIR, λ=3-25 μm) light has been used as animaging technique for a variety of applications, including medicaldiagnosis, night vision, and remote sensing. Mid-IR imaging providessome advantages over other IR, visible, and/or ultra-violet (UV)light-based imaging. Many substances absorb mid-IR light and causeresonant vibrational excitations of chemical bonds, which makes it anearly universal label-free tool applicable to almost any material.Additionally, different compounds exhibit unique absorption features inthe mid-IR spectrum, enabling mid-IR light to be used in spectroscopicapplications. Mid-IR spectroscopy can be used to identify andcharacterize the chemical composition of objects. Additionally, mid-IRspectroscopy can be used to track activity of biological systems.

Mid-IR light also experiences significantly less scattering than visibleor UV light, allowing it to penetrate much farther into scatteringmaterials. Such reduced scattering can be useful when conductingmeasurements over a long distance, such as in gas sensing and remotesensing. Additionally, since any object that emits heat—at least abovethe temperature of absolute zero—radiates infrared light, mid-IR imagingcan also be achieved without a separate illumination source. The objectbeing imaged can itself serve as a light source and thus enable“passive-detection.” Further, since the photon energy of MIR radiationis relatively low, no photochemical reactions are stimulated, allowingmid-IR imaging to be non-disruptive. Other biological system imagingtechniques, such as fluorescence imaging, suffer from photo-bleachingand phototoxicity effects.

Although mid-IR imaging can be a powerful tool, there are some drawbacksinherent to the physical nature of mid-IR light and mid-IR devices. Onedifficulty in mid-IR imaging is the scarcity of the infrared detectors.There are two major types of infrared detectors: thermal detectors andphotonic detectors. Thermal detectors are typically affordable, but slowand sometimes inaccurate. Photonic detectors, on the other hand, havebetter read out speed and accuracy compared to thermal detectors, butare usually expensive and require an extremely low temperature tofunction (e.g. −203° C.). Commonly used indium antimonide (InSb) ormercury cadmium telluride (MCT) detectors usually requires a liquidnitrogen (LN₂) or thermoelectric (TE) cooling system, which make theminconvenient to use and economically undesired.

Typical mid-infrared imaging techniques also tend to have limitedspatial resolution, due to the Abbe diffraction limit. The spatialresolution of a specific color of light is linearly dependent on itswavelength, such that longer wavelengths lead to lower spatialresolution. Mid-IR light includes wavelengths from 3 to 25 μm, whilevisible light spans from 390 nm to 700 nm. This means that, in typicalimaging systems, the spatial resolution achievable from mid-IR light canbe anywhere from 5 to 65 times lower than that of visible light. Thismeans that for biological systems, conventional MIR imaging typicallyprovides access to spatial information down to the tissue level. Oneimportant application at this level known as “thermography” can be used,for example, as a diagnosis tool for early breast cancer detection.However, analysis of nano-scale structures is usually not possible withconventional MIR techniques. Due to these physical limitations, imagingsubmicron-sized objects using mid-IR light has been infeasible inexisting imaging systems.

The apparent problem of low resolution in mid-IR imaging has beenapproached using different technologies, such as Scanning ProbeMicroscopy (SPM), solid-immersion lens, scattering-type scanningnear-field optical microscopes (s-SNOM, a technique that utilizes ametalized atomic force microscope tip to scatter broadband infraredradiation), and others. However, those methods rely heavily onintegration of sophisticated instruments and they deprive MIRmeasurement of its potential to be performed in media. At the same time,the cumbersome (cooling needed), expensive and less reliable MIRdetectors are in the standard configuration of those methods.

Additionally, Fourier transform spectroscopy of the scattered lightgives information about molecular vibrations with a spatial resolutionas high as 20 nm. Another alternative form of SPM integration with MIRimaging relies on photothermal induced resonance effects. In thismethod, pulsed MIR light is used in total internal reflection with aZnSe crystal. Absorption of the MIR light by the material in contactwith the prism causes thermal mechanical expansion, which is detected bythe SPM tip. Scanning the tip over the sample yields super-resolved MIRimages. Also, in order to tackle the limited resolution, solid immersionlens is a viable approach too. MIR radiation is focused by the solidimmersion lens into a material with a high refractive index, whichthereby results in a reduced focal spot by a factor of the refractiveindex, comparing to that in vacuum. The evanescent wave escapes from thehigh refractive index material into the air, and hence probes the sampleon the surface in a near field mode.

Although advances have been made with the methods described above, thosetechniques rely heavily upon the integration of sophisticatedinstrumentations, which results in complex experimental setups. Also,none of the approaches above overcomes the difficulty of detectorsensitivity. A traditional expensive LN₂- or TE-cooled detector is usedwithin the standard configuration of those experimental setups. Thus, amethod that not only improves the spatial resolution of MIR imaging, butalso circumvents the usage of a standard MIR detector, is desired.

It is accordingly an objective of the present invention to providemid-IR imaging systems and methods—which utilize the spectroscopicbenefits of mid-IR—while also improving upon the spatial resolutionlimitations inherent in typical mid-IR imaging systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an example photothermal imaging system,according to an example embodiment.

FIG. 2A is an image of polystyrene beads in water captured by theexample photothermal imaging system using a mid-IR pump beam wavelengthof 2850 cm⁻¹.

FIG. 2B is an image of polystyrene beads in water captured by theexample photothermal imaging system using a mid-IR pump beam wavelengthof 3300 cm⁻¹.

FIG. 3A is a MIR-PHI image of a polystyrene bead in glycerin captured bythe example photothermal imaging system.

FIG. 3B is a line-profile of the MIR-PHI image of FIG. 3A across thepolystyrene bead.

FIG. 4 is an image of polystyrene beads in air on a glass coverslipcaptured by the example photothermal imaging system.

FIG. 5A is a MIR-PHI spectrum graph of a polystyrene bead on a glasscoverslip captured by the example photothermal imaging system.

FIG. 5B is an ensemble Fourier transform infrared measurement (FT-IR)the polystyrene bead on a glass coverslip from FIG. 5A.

FIG. 6A is a MIR-PHI measurement of a polystyrene bead in carbondisulfide in a first irradiance configuration, captured by the examplephotothermal imaging system.

FIG. 6B is a MIR-PHI measurement of the polystyrene bead in carbondisulfide in a second irradiance configuration, captured by the examplephotothermal imaging system.

FIG. 6C is a MIR-PHI measurement of the polystyrene bead in carbondisulfide in a third irradiance configuration, captured by the examplephotothermal imaging system.

FIG. 6D is a MIR-PHI measurement of the polystyrene bead in carbondisulfide in a fourth irradiance configuration, captured by the examplephotothermal imaging system.

FIG. 7A is a MIR-PHI image of a polystyrene bead in glycerin, capturedby the example photothermal imaging system.

FIG. 7B is a MIR-PHI line-profile graph of a polystyrene bead inglycerin, captured by the example photothermal imaging.

DETAILED DESCRIPTION

The following description of example methods and apparatus is notintended to limit the scope of the description to the precise form orforms detailed herein. Instead the following description is intended tobe illustrative so that others may follow its teachings.

Mid-infrared radiation is resonant to the vibrational excitations ofmany chemical bonds and can, therefore, its absorption spectra reveal awealth of information of molecular composition. When MIR spectroscopy isperformed in a localized area, identifying, characterizing and trackingmicroscopic objects become accessible. Databases of MIR spectra enableanalysis of complex structures, such as single live cells and individualprotein complexes. Since MIR spectroscopy reflects the vibrations ofchemical bonds, the unique combination of different chemical bonds in amolecule gives rise to a characteristic spectral “fingerprint.” Thiscircumvents the common procedures of applying stains, fixatives, orexogenous fluorophores for imaging biological samples.

Although MIR spectroscopy is a powerful tool, there are two majorinherent drawbacks. First, MIR imaging typically has a limited spatialresolution. According to Abbe diffraction, the minimum spatialresolution that can be achieved is

$\begin{matrix}{{resolution} = \frac{\lambda}{2*{NA}}} & (1)\end{matrix}$

where λ denotes the wavelength of light and NA denotes the numericalaperture of the objective. Thus, MIR radiation typically has a spatialresolution that is around an order-of-magnitude worse than visiblelight, given the same or similar optics conditions.

In order to solve the problems described above, a technique that uses anaffordable and reliable detector and has a superior spatial resolutionis needed. Mid-infrared photothermal heterodyne imaging (MIR-PHI)systems overcome the diffraction limit of traditional MIR imaging.Photothermal imaging systems of the present application utilize acombination of a mid-IR “pump” laser and a visible light “probe” laser.Under one theory of operation, irradiating a sample with the pump lasermay heat the sample and its surrounding medium, which can cause athermal “lens” to form.

The photothermal effect is the phenomenon that, when an object absorbsphotons, it can release the energy through radiating heat to itssurrounding medium. This forms a temperature gradient in the mediumaround the object. Then by using a separate probe light source with amuch shorter wavelength than the MIR wavelengths (i.e., at a visible ornear-IR wavelengths), the MIR-PHI can measure mid-IR absorption of thesample with a spatial resolution well below the Abbe diffraction limitof the mid-IR pump beam. The use of visible or near-IR wavelength forthe probe beam enables use of visible/near IR photodiodes as detectorsdisclosed herein that may accomplish using reliable and cost-effectivedetectors with higher sensitivity than mid-IR detectors. Since therefractive index of the medium is related to its temperature, thetemperature gradient creates a refractive index gradient which resemblesa lens. This thermal lens can then affect the propagation of a probebeam and thus generate a photothermal signal. Raster scanning in twodimensions—using a movable platform such as a piezo stage—can create acontrast between the presence and absence of the thermal lens, therebyforming a photothermal image.

According to one embodiment, a pump beam is at least partially absorbedby an analyte or sample, which gives rise to localized heating. Theheating effect can cause the refractive index of the particle and itssurrounding environment to change, creating a refractive index gradient.This refractive index gradient in the medium behaves in a similar way asa lens, and is referred to as a “thermal lens.” The probe beam—which ispreferably non-resonant to the analyte's absorption—is then used todetect the thermal lens effect. A shorter wavelength probe can beutilized, so that the Abbe diffraction resolution is much improved. Insome embodiments the probe wavelength can be selected in the visibleregion, where reliable, sensitive and affordable detectors are readilyavailable. Furthermore, the power of the probe can be operated at ahigher power levels, in order to minimize the photon noise, as long asthe power level is below the sample damage threshold.

Photothermal imaging offer several advantages that solve the problemsassociated with conventional mid-IR imaging. For the scarcity ofdetector issue, since the photothermal effect a visible or near-infraredlight source, such as a visible or near IR laser can be used as theprobe. Detectors for visible or near-infrared light can be faster,cheaper and more sensitive. Also, the spatial resolution of photothermalimaging is determined by the probe wavelength (not the wavelength of themid-IR heating beam), which implies a significant improvement over thespatial resolution of conventional mid-IR imaging. This can be veryuseful in many applications, for instance in the medical imaging field,because it can potentially push the limit of mid-IR imaging to asubcellular level or even smaller, while the commonly used scale rightnow is at the level of imaging tissues. On the other hand, using amid-IR pump beam in the photothermal technique also remedies one of theshortcomings of conventional photothermal imaging measurements. Sincethe photothermal effect is based on the absorption of photons, theconventional photothermal imaging measurements using visible or near-IRpump lasers are limited to studying materials such as noble metalnanoparticles and dye molecules that absorb in the visible region. Sincealmost all compounds absorb mid-IR light and then generate thermalenergy, the applicability of photothermal imaging is vastly broadened bythe introduction of the mid-IR technique.

In this disclosure, PHI experiments are described that utilize a mid-IRpump laser source. It is shown that MIR-PHI imaging systems can achievemicrometer spatial resolution, which is much smaller than thediffraction limit of the mid-IR light source. The combination of mid-IRand photothermal imaging techniques exploits affords the benefits ofimproved spatial resolution with robust and inexpensive detectors, whilealso maintaining the spectroscopic potential associated with mid-IRabsorption.

In summary, MIR-PHI systems overcome the diffraction limit typicallyinherent to MIR imaging, while using fast and accurate visible/near IRphoto detectors. With our counter-propagating geometry, it is possibleto obtain high contrast images of 1.1 μm polystyrene beads in glycerinand in air. Clear absorption spectra were also acquired locally on thesingle particles. These improvements to mid-IR imaging have thepotential to provide opportunities for researchers to investigatebiological systems in media on a spatial scale that was not previouslyachievable, and thus has broad application potential in biologicalimaging.

FIG. 1 is a schematic diagram of an example photothermal imaging system100. In photothermal imaging system 100, a probe laser 110 emits a probebeam 111 toward a probe mirror 112, which reflects the probe beam 111onto beam splitter 113. Beam splitter 113 may be a polarizingbeamsplitter or similar to allow the probe beam 111 reflecting off theprobe mirror 112 to pass through to the objective 114 and onto a firstside of sample 130. In some implementations, the probe beam 111 is avisible light laser beam, and/or a near IR laser beam.

The pump laser 150 emits a pump beam 151 toward a pump mirror 152, whichreflects pump beam 151 toward a reflective objective 153 The reflectiveobjective 153 may serve to irradiate the sample 130 from a second side,which is opposite to the first side. This arrangement—in which the probebeam 111 and pump beam 151 illuminate or irradiate the sample 130 fromopposite sides—may be referred to herein as a “counter propagatinggeometry.” In some implementations, the pump beam 151 may be a mid-IRlaser beam.

During operation, the pump beam 151 irradiates the sample 130, whichabsorbs the mid-IR light, causing localized heating on the sample 130.This localized heating can lead to the thermal lens effect describedherein. The probe beam 111 incident on the sample 130 interacts with aregion of the sample illuminated by the pump beam. which scatters someof the light of probe beam 111, for example due to the thermal lenstemperature gradient of an absorbing region of the sample Some of thereflected probe beam 111 then travels back toward the beam splitter 113,which directs the reflected probe beam 111 toward a photodiode 122. Thephotodiode 122 then collects and measures the probe beam light 111,which may be stored on a computing device or the like.

The step of mid-IR irradiation, and probe beam reflection measurement bythe photodiode 122 may be repeated at different locations on sample 130.Movable platform 140—which may be a base controlled by servos, a piezostage, or other actuating platform—may move the sample 130 laterally orlongitudinally (with respect to the objective 114) in order to positionobjective 114 and reflective objective 153 over a different location onsample 130. That new location may then be irradiated by the mid-IR pumpbeam 151, and measured by the reflected probe beam 111 incident onphotodiode 122. By repeating this step at various locations of thesample 130 and storing the read out values from photodiode 122, acomputing device may then construct a 2D image representing thephotothermal intensities of the sample.

The photodiode 122 may provide a probe signal input indicative of theintensity of the reflected probe beam 111 to a lock-in amplifier 120.The lock-in amplifier 120 may be configured on the basis of thewavelength of the probe beam 111 (i.e., referenced to the modulationfrequency of the pump beam) and may be capable of detecting minorfluctuations in the properties of the probe beam 111 caused by thethermal lens effect of the sample 130. The lock-in amplifier 120 mayalso receive a pump beam modulation signal 123 from the pump laser 150indicative of the modulation frequency of the pump beam 151 (e.g., 100kHz, 150 kHz, etc.). In some implementations, the lock-in amplifier 120also receives a signal from the probe laser 110 indicative of thefrequency or wavelength of the probe beam 111.

An example MIR-PHI system (such as the photothermal imaging system 100shown in FIG. 1) comprises two laser sources. A tunable MIR pump laser150 (e.g., with wavelengths between 2.5-3.7 μm), operating under pulsemode at 150 kHz, is used as the pump laser source. This range covers theC—H, N—H, and O—H stretch absorption peaks, which are present in organiccompounds. A tunable pump light source enables the measurement to bemade a multiple mid-IR wavelengths, thereby allowing for data to betaken that is representative of an IR absorption spectrum at sub-micronspatial resolution. For some of the experiments described below, thepump laser is tuned to 3030 cm⁻¹ to excite 1.1 μm polystyrene beads. Acounter-propagating, continuous wave (CW) 532 nm probe laser 110 is usedto detect the thermal lens created by the pump laser 150. The pump laser150 is focused by a reflective objective 153 (NA=0.65), and the probebeam 111 is focused by a long working distance, high NA glass objective114 (NA=0.80). This arrangement is different to that used in previousMIR-PHI measurements, where a single reflective objective was used tofocus the pump and the probe at the sample. The advantage of this setupis that a tighter focus can be obtained for the probe beam 111, yieldinghigher spatial resolution. A disadvantage is that twocounter-propagating beams have to be carefully aligned with each otherto maximized the PHI signal. The reflected probe beam is detected by aSi-photodiode, and the signal from the Si-photodiode is sent to alock-in amplifier (e.g., a Stanford Research SR-844 RF Lock-inAmplifier), which is triggered by pulses from the MIR laser at 150 kHz.Raster scanning with a piezo stage 140 across an area on the order of10×10 μm therefore results in a MIR-PHI image.

In terms of the sample preparation procedure, an optical cell with highmid-infrared transmission (e.g., two sapphire windows) can be used.Specimens are applied on a surface of one of the sapphire windows bydrop-casting or spin-coating. A small amount of medium can be droppedonto the specimen. Parafilm or a sealant can then be used to seal theperipheral areas of the two windows together, and thus create an opticalcell with a thin chamber of the specimen surrounded by the solvent. Thethickness of the sapphire window was selected to be small, so that itwill be within the working distance of the objective.

The probe beam 111, which can be a visible or near-infrared laser, maybe operated in the continuous wave (CW) mode. In one embodiment, the twolasers may be designed to be counter-propagating whereby the sample isilluminated from different sides by the two lasers. This design avoidsthe usage of dichroic mirrors for combining the mid-IR and visiblelight, which may be desirable due to the dichroic mirrors for these twolaser wavelengths vastly attenuating the intensity of the mid-IR beam.This arrangement also allows the use of high magnification microscopeobjectives for the probe beam 111 (since objectives that transmit IRlight are typically low magnification).

In one embodiment, the probe beam 111 is directed at a first samplesurface while the pump beam 151 is directed to a second sample surfaceopposite the first. In other embodiments, the surfaces illuminated bythe two beams may not be exactly opposite one another. In one instance,the pump beam 151 may be directed to an adjacent surface as the onebeing illuminated by the probe beam 111. In another embodiment, the pumpbeam 151 may be directed to the same area where the surface illuminatedby the probe beam 111 resides. In one embodiment, the angle ofseparation between the pump beam 151 and the probe beam 111 may be about180 degrees. In other embodiments, the angle of separation may be someangle less than 180 degrees but sufficient to provide a separate pathwayfor the pump beam 151 and probe beam 111. The angle of separationprovides two pathways, one for the probe beam 111 and one for the pumpbeam 151 to illuminate the same area of the sample under test.

In one embodiment, the reflected probe beam from the sample surface isthen collected by the same objective used to focus it, and detected by aphoto detector, which can simply be a regular visible light photodiode(e.g., Si, InGaAs, Ge, etc.). The signal from the photo detector 122 isthen processed by a lock-in amplifier 120, referenced to the modulationfrequency (via signal 123) of the pump beam 151. This allows the lock-inamplifier 120 to detect the amplitude and the relative phase of thephotothermal signal output from the photodiode 122. A raster scan of thesample over a certain area, which can be conducted by a piezo-stage 140,then creates a mid-IR photothermal image.

Signal processing devices, such as the lock-in amplifier, may sample ormeasure one or more property of collected probe light (i.e., reflectingoff a sample or a region of a sample). For instance, a signal processingdevice may—based on parameters of the photothermal imaging system (e.g.,a pump beam pulse or modulation frequency, a wavelength of the pumpbeam, a wavelength of the probe beam, etc.)—determine a frequency, shiftin frequency, phase, phase shift, intensity, or any other property ofthe collected probe beam light. The property or properties measuredand/or determined by the signal processing device may serve as a basisfor analyzing, imaging, conducting spectroscopic analysis, and/or otheroperations on a sample or a region of a sample.

In order to obtain good imaging contrast, the selection of themid-infrared wavelength is important. If the analyte has an absorptionpeak at a certain wavelength while the solvent doesn't absorb at thatsame wavelength, the contrast of the image will be high. This can beachieved by using solvents with limited mid-infrared absorptionfeatures, such as CS₂ and CCl₄. For water—which has a very broadabsorption in the mid-IR region—two strategies can be used to obtain animage with good contrast. First, images can be captured at a wavelengthwhere the absorption of water is limited and the absorption from theanalyte is at its maximum. For example, at 2850 cm⁻¹, a polystyrene beadcan be clearly imaged with the mid-PHI technique in water (as shown inFIG. 2A). Another method involves tuning the mid-IR wavelength towater's absorption peak while the analyte doesn't have any absorption.For example, at 3300 cm⁻¹, a clear image can also be collected forpolystyrene beads in water (shown in FIG. 2B). The two images of thesame object using these two strategies will be complementary with eachother, one with a dark background and a bright signal, and the other onewith a bright background and a dark signal.

The solvent used in the photothermal imaging system can also affect itsperformance. Some organic solvents have outstanding photothermalproperties, such as CS₂ and CCl₄. Photothermal imaging with thesesolvents results in a very high signal-to-noise sensitivity. However,they are usually toxic, volatile, and not compatible with biologicalsamples. Glycerin, which is biocompatible and viscous, has a relativelyhigh photothermal performance. Water, as the ideal medium for biologicalsamples, does not have a sensitive photothermal response compared toother solvents. However, water was used to achieve a visualization ofone micron polystyrene particles in water at a single particle levelusing this technique. For example, 1.1 μm polystyrene beads weresuccessfully visualized with super-resolution in water (as shown in FIG.2A and FIG. 2B).

The selection of medium is an important factor for PHI measurements. Apreferable medium for PHI includes the following characteristics. First,a large refractive index provides for better performance of the thermallens. Second, a preferable PHI medium has a small heat capacity, withthe aim to maximize the change in temperature created by a given amountof heat transferred from the particle. Additionally, the more sensitivethe refractive index is to temperature, the shaper the refractive indexgradient will be in the thermal lens. A figure-of-merit for the overallphotothermal performance of a medium can be expressed with equation (2)below:

$\begin{matrix}{{\sum{Photothermal}} = {n{\frac{\delta \; n}{\delta \; T}}\left( \frac{1}{C_{T}} \right)}} & (2)\end{matrix}$

where n denotes the refractive index, T denotes the temperature, andC_(T) represents the heat capacity under temperature T. Researchers havecompared the performance of different common solvents. Among them,carbon disulfide (CS₂) was found to have preferable photothermalproperties. Additionally, carbon tetrachloride, chloroform, hexane,decane, ethanol, glycerin, and water are other solvents that exhibitpreferable photothermal properties.

The thermal diffusion length is another factor that has a large impacton PHI image contrast. Since the particles are heated by the pump beamintermittently at the modulation or pulse frequency, the magnitude ofthe frequency is directly related to the thermal diffusion length. Lowerfrequencies give longer thermal diffusion lengths and yield higherphotothermal signals, until the thermal diffusion length exceeds thesize of the focal spot. Therefore a focal spot size that matches thethermal diffusion length will be optimal. The thermal diffusion length(r_(th)) of the medium can be calculated with the equation (3) below:

$\begin{matrix}{r_{th}^{2} = {\frac{2D_{th}}{\Omega} = \frac{2k}{C_{p}\Omega}}} & (3)\end{matrix}$

where D_(th) is the thermal diffusivity, k is the thermal conductivityof the medium, Ω is the modulation frequency, and C_(p) is the heatcapacity per unit volume photothermal medium. At the same time, thelower the modulation frequency, the longer the thermal diffusion length,and the worse the spatial resolution will be. That is why we triggeredour measurement at the high pulse frequency of 150 kHz, which is muchhigher than that in the previous literatures (20 Hz). This madesub-diffraction limit resolution (of the pump beam) practicallyplausible. The thermal diffusion length (r_(th)) of different media arecalculated. Under 150 kHz of modulation, carbon disulfide (CS₂) has adiffusion length of 1.3 μm, that of glycerin is 1.2 μm, and that ofwater is 1.3 μm. A 532 nm probe laser, the size of the focal spot isestimated to be about 0.9 μm for the 0.8 NA objective used in ourexperiments, which roughly matches the thermal diffusion lengths ofdifferent media.

In a MIR-PHI setup, a MIR laser is used as a pump beam to createabsorption events on sample objects. A temperature gradient is therebycreated around the heated particles. Since the refractive index of themedium is directly related to its temperature, a temperature gradientwill result in a refractive index gradient, which can then be detectedby another probe beam. The information of the absorption event istherefore measured indirectly by the change in the probe light. Sincethe light being measured is the probe (usually visible light), it is nolonger constrained by the Abbe diffraction limit of the MIR, and onlydetectors for visible light are needed.

The integration time on the lock-in of the lock-in amplifier 120 is animportant factor in system performance as well. Generally, the longerthe integration time, the better the signal-to-noise for the PHI signal.However, extended lock-in integration time substantially deterioratesMIR-PHI's real-time detection capability. In experiments describedherein, 300 ms is used under CS₂ medium, and 1 second is used forglycerin and water.

FIGS. 2A and 2B are images of polystyrene beads in water captured by theexample photothermal imaging system using a 532 nm probe laser. In image200, the mid-IR pump beam is configured to operate at a wavelength of2850 cm⁻¹. In image 250, the mid-IR pump beam is configured to operateat a wavelength of 3300 cm⁻¹.

These two wavelengths were selected to form the largest contrast betweentwo materials. At 3300 cm⁻¹, water has a high absorption strength, whilethe absorption from polystyrene should be low. On the other hand, at2850 cm⁻¹, polystyrene has a local maxima in its mid-IR absorptionspectra, while the absorption from water falls to around 20%. Hence, thetwo chosen wavelengths 3300 cm⁻¹ and 2850 cm⁻¹ should form complementaryimages with relatively high contrasts.

The results, as shown in FIGS. 2A and 2B, confirmed the aboveprediction. In FIG. 2A, the polystyrene beads appear to have a negativephotothermal signal (lower than the background). In contrast, in FIG.2B, the polystyrene beads appear positive (higher than the background).The S/N ratio measured at 3300 cm⁻¹ is 4, while at 2850 cm⁻¹ it becomesaround 11.

Post-processing of the resulting images can be done to improve the S/Nlevel. For example, image fusion can be a viable method to enhance imagecontrast. However, in this example, a specific algorithm could be usedto properly combine the two images, in order to retain the appropriatechemical information.

FIG. 3A is a MIR-PHI image 300 of a polystyrene bead in glycerincaptured by the example photothermal imaging system. As shown in FIG.3A, when a polystyrene bead is placed in glycerin, strong MIR-PHI signalwas obtained (S/N>100) within a short acquisition time. By fitting thesignal intensity profile into a Gaussian beam profile, a Gaussian beamwaist of 0.62 μm±0.01 μm was measured, which is much smaller than theAbbe diffraction limit of the MIR light (around 2.33 μm). Thus,super-resolution was experimentally achieved.

FIG. 3B is a line-profile graph 350 of the MIR-PHI image of FIG. 3Aacross the polystyrene bead. The line-profile graph 350 may be used tocalculate performance factors, such as signal-to-noise ratio (SNR). Asshown in FIG. 3B, the photothermal imaging system achieved an SNR of106, with a Gaussian beam waist of approximately 0.62 μm, for apolystyrene bead in glycerin. This image demonstrates for example theability MIR-PHI embodiments described in this disclosure to achievespatially resolved measurements of IR absorption with sub-micron spatialresolution.

FIG. 4 is an image 400 of polystyrene beads in air on a glass coverslipcaptured by the example photothermal imaging system. In FIG. 4, barepolystyrene beads were not immersed in any liquid media, but simplydrop-cast on a glass coverslip. While maintaining at a high contrast,the shape of the MIR-PHI signal is not perfectly spherical, which may bedue to a slight misalignment in the optical system. The MIR-PHI signalmay be improved by implementing a movable piezo z-axis objective mount,and finer micrometer actuators for positioning.

FIG. 5A is a MIR-PHI spectrum graph 500 of a polystyrene bead on a glasscoverslip captured by the example photothermal imaging system. When boththe pump beam and the probe beam rest on one particle, tuning the pumpbeam (MIR) can provide local absorption spectral information. TheMIR-PHI signal can be measured at a plurality of wavelengths of the pumpbeam, resulting in a clear MIR absorption spectrum of PS beads as shownin FIG. 5A.

FIG. 5B is an ensemble Fourier transform infrared measurement (FT-IR)550 the polystyrene bead on a glass coverslip from FIG. 5A. The MIR-PHIspectrum graph 500 of FIG. 5A roughly matches with the ensemble FT-IRmeasurement of the polystyrene beads sample in FIG. 5B. Slightdifferences in peak positions are due to the mismatch in scanningspectral step-size (4 cm⁻¹ vs 2 cm⁻¹).

Although the peaks in FIG. 5A and FIG. 5B are not precisely identical,the two graphs illustrate that a MIR-PHI spectrum can serve to identifythe chemical composition of a sample. In this example, the MIR-PHIspectrum peaks in FIG. 5A correspond with spectrum dips in the FT-IRspectrum in FIG. 5B. Given a database of known FT-IR spectra, theMIR-PHI spectrum of some sample may be compared against that database toidentify the chemical composition of that sample.

FIGS. 6A-6D are MIR-PHI measurements of a polystyrene bead in carbondisulfide (CS₂) at different irradiance configurations, captured by theexample photothermal imaging system. In FIG. 6A, image 600 andline-profile graph 605 are MIR-PHI measurements taken with a radiancelevel of 0.1 Watts, resulting in a Gaussian beam waist of 0.85 μm and asignal-to-noise ratio (SNR) of 19. In FIG. 6B, image 610 andline-profile graph 615 are MIR-PHI measurements taken with a radiancelevel of 0.35 Watts, resulting in a Gaussian beam waist of 0.94 μm andan SNR of 24. In FIG. 6C, image 620 and line-profile graph 625 areMIR-PHI measurements taken with a radiance level of 0.5 Watts, resultingin a Gaussian beam waist of 1.02 μm and an SNR of 28. In FIG. 6D, image630 and line-profile graph 635 are MIR-PHI measurements taken with aradiance level of 0.6 Watts, resulting in a Gaussian beam waist of 1.03μm and an SNR of 19.

FIGS. 6A-6D were produced as a part of a probe irradiance dependencestudy, which was performed to investigate the optimal probe irradiancelevels. An area of 15×15 μm is studied (scanning step size: 0.5 μm),with single 1.1 μm polystyrene beads present in the field of view.

Comparison between FIGS. 6A, 6B, and 6C clearly shows an enhancement ofthe signal level with an increase in the probe irradiance. However,comparing the results between FIG. 6C and FIG. 6D displays an oppositetrend, where a radiance level increase caused a decrease in signallevel. Possible sample damage may explain the signal degradation betweenFIG. 6C and FIG. 6D. In this example, probe irradiance around 0.4 W wasdetermined to be preferred for this system.

The line profiles in the graphs of FIGS. 6A-6D were used to estimate thespatial resolution. The results show that the beam waist is around 1 μm,very similar to the size of the polystyrene beads. On the other hand,the theoretical Abbe diffraction limit of the pump beam is

$\frac{\lambda}{2*{NA}} = {2.33\mspace{14mu} {{\mu m}.}}$

Thus, super-resolution MIR imaging was achieved.

Although some nonpolar media, such as CS₂ and CCl₄ have excellentphotothermal properties and minimum infrared absorption, their lowboiling points and poor biocompatibility substantially lessen theirpotential for applications. Bio-compatible media, such as glycerin andwater, are of much greater interest. Hence, it is worthwhile to test thephotothermal imaging system with the 1.1 μm polystyrene beads in bothglycerin and water.

FIG. 7A is a MIR-PHI image 700 of a polystyrene bead in glycerin,captured by the example photothermal imaging system. FIG. 7B is aMIR-PHI line-profile graph 750 of the MIR-PHI image 700 shown in FIG.7A. Glycerin, although not as ideal as CS₂ and CCl₄ for a photothermalmedium, is still around 5 times better than water for this application.In FIG. 7, a single 1.1 μm polystyrene bead is imaged with much smallerstep size (0.2 μm). However, the signal-to-noise ratio falls to theorder of 11, which makes this measurement more difficult. The reason forthe inferior S/N ratio lies in the fact that the mid-IR absorptions ofGlycerin and polystyrene are largely overlapped in this region.Therefore the background signal level for the PHI image is much higherthan that from CS₂, which in turn gives rise to the deteriorated S/Nratio.

This disadvantage was believed to be even more severe for measurementsin water, because of the broad absorption band that water has in themid-IR region. In order to alleviate this issue, the absorption of thepolystyrene and water were carefully compared. The tests and results ofthe MIR-PHI imaging of polystyrene beads in water is described above inrelation to FIGS. 2A and 2B.

In summary, MIR-PHI not only significantly improves the spatialresolution of traditional MIR imaging, but also circumvents the usage ofa sophisticated mid-IR detector. It therefore provides a robustsuper-resolution MIR imaging solution in an inexpensive way. Theexperimental results show that measurements in CS₂ imaged 1.1 μmpolystyrene beads at a reasonable S/N ratio, with super-resolutioncapabilities. Results in glycerin and water, although displaying similarresolution, have much poorer S/N ratios. Hyperspectral imaging will beperformed in the near future to exploit the wealth of chemicalinformation in the mid-IR region. Also, fine tuning in the z axis of theimaging plane can be a direction to significantly improve thesignal-to-noise ratio. Image post-processing with image fusion or othermethods can potentially enhance the imaging contrast too.

Although certain example methods and apparatus have been describedherein, the scope of coverage of this patent is not limited thereto. Onthe contrary, this patent covers all methods, apparatus, and articles ofmanufacture fairly falling within the scope of the appended claimseither literally or under the doctrine of equivalents.

It should be understood that arrangements described herein are forpurposes of example only. As such, those skilled in the art willappreciate that other arrangements and other elements (e.g., machines,interfaces, operations, orders, and groupings of operations, etc.) canbe used instead, and some elements may be omitted altogether accordingto the desired results. Further, many of the elements that are describedare functional entities that may be implemented as discrete ordistributed components or in conjunction with other components, in anysuitable combination and location, or other structural elementsdescribed as independent structures may be combined.

While various aspects and implementations have been disclosed herein,other aspects and implementations will be apparent to those skilled inthe art. The various aspects and implementations disclosed herein arefor purposes of illustration and are not intended to be limiting, withthe true scope being indicated by the following claims, along with thefull scope of equivalents to which such claims are entitled. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular implementations only, and is not intended to belimiting.

We claim:
 1. A method for detecting infrared light absorption in asample with submicron resolution, the method comprising the steps: a)illuminating a region of the sample with a pump beam generated by afirst light source, wherein at least a portion of the pump beam isabsorbed by the region of the sample; b) illuminating the region of thesample with a probe beam generated by a second light source, wherein theprobe beam has a first wavelength; c) collecting, by a detector, aportion of the probe beam coming from the region of the sample; and d)analyzing the collected light to construct a signal indicative ofinfrared absorption by the region of the sample with submicron spatialresolution.
 2. The method according to claim 1, wherein analyzing thecollected light comprises the steps: generating, based on the collectedprobe light and the first wavelength, a signal indicative of the portionof the pump beam absorbed by the region of the sample, wherein thesignal represents an amount of infrared light absorbed by the region ofthe sample.
 3. The method according to claim 1, further comprising thestep of repeating steps (a)-(d) at plurality of regions on the sample toconstruct a map of the signal indicative of infrared absorption with aspatial resolution of less than 1 micron.
 4. The method according toclaim 1, wherein illuminating the region of the sample with the pumpbeam comprises illuminating a first side of the region of the samplewith the pump beam, wherein illuminating the region of the sample withthe probe beam comprises illuminating a second side of the region of thesample with the probe beam, and wherein the first side of the region ofthe sample is opposite to the second side of the region of the sample.5. The method according to claim 1, wherein the pump beam travels alonga first pathway toward the region of the sample, wherein the probe beamtravels along a second pathway toward the region of the sample, andwherein the first pathway does not intersect with the second pathway. 6.The method according to claim 1, wherein the pump beam is directedtoward a first sample surface, wherein the probe beam is directed towarda second sample surface, and wherein the first sample surface isadjacent to the second sample surface.
 7. The method according to claim1, wherein the pump beam is directed toward a first sample surface,wherein the probe beam is directed toward a second sample surface, andwherein the first sample surface is the same as the second samplesurface.
 8. The method according to claim 1, further comprising thestep: modulating the pump beam according to a first modulationfrequency.
 9. The method according to claim 8, wherein the firstmodulation frequency is greater than or equal to 100 kHz.
 10. The methodaccording to claim 1, wherein the first light source is a mid-infraredlaser having at least one emission wavelength within the range of 3 to25 micrometers.
 11. The method according to claim 1, wherein the secondlight source is a visible light laser having at least one emissionwavelength that is less than or equal to 800 nanometers.
 12. The methodaccording to claim 1, further comprising the step: focusing, by areflective objective, the pump beam onto the region of the sample. 13.The method according to claim 1, further comprising the step: focusing,by a refractive objective, the probe beam onto the region of the sample.14. The method according to claim 1, wherein the analyzing stepcomprises: sending a signal from the detector to a lock-in amplifierconfigured to determine at least one of (i) an amplitude and (ii) aphase of the probe beam reflecting from the region of the sample;providing the lock-in amplifier a reference signal to a modulationfrequency of the pump laser; and using the lock-in amplifier toconstruct the signal indicative of infrared absorption by the region ofthe sample.
 15. The method according to claim 1, wherein the sample isheld by a cell adapted for high infrared transmission, wherein the cellincludes sapphire windows.
 16. The method according to claim 1, whereinabsorption of infrared radiation from the pump beam by the sampleresults in a temperature increase of the region of the sample andwherein propagation of the probe beam is affected by a thermal lensformed by the temperature increase.
 17. A system for detecting infraredlight absorption in a sample with submicron resolution, the systemcomprising: a first light source operable to illuminate a region of thesample with a pump beam of mid infrared radiation, wherein at least aportion of the pump beam is absorbed by the region of the sample; asecond light source operable to illuminate the region of the sample witha probe beam; a detector operable to collect and measure at least aportion of the probe beam coming from the region of the sample; and asignal processing device configured to generate, based on the probelight collected by the detector, a signal indicative of an amount ofinfrared light absorbed by the region of the sample, with sub-micronspatial resolution,
 18. The system according to claim 17, wherein thesystem further comprises: a movable platform coupled to the sample andoperable to move the sample in two dimensions with respect to the firstlight source and the second light source to illuminate a plurality ofregions of the sample with the pump beam and probe beam; and wherein thesignal processing unit generates the signal indicative of infraredabsorption of light by the sample at the plurality of regions of thesample to generate an image indicated of infrared absorption of thesample.
 19. The system according to claim 17, wherein the first lightsource is arranged to illuminate a first side of the region of thesample with the pump beam, wherein the second light source is arrangedto illuminate a second side of the region of the sample with the probebeam, and wherein the first side of the region of the sample is oppositeto the second side of the region of the sample.
 20. The system accordingto claim 17, wherein the first light source is arranged to emit the pumpbeam that travels along a first pathway toward the region of the sample,wherein the second light source is arranged to emit the pump beam thattravels along a second pathway toward the region of the sample, andwherein the first pathway does not intersect with the second pathway.21. The system according to claim 17, wherein the first light source isarranged to emit the pump beam toward a first sample surface, whereinthe second light source is arranged to emit the pump beam toward asecond sample surface, and wherein the first sample surface is adjacentto the second sample surface.
 22. The system according to claim 17,wherein the first light source is arranged to emit the pump beam towarda first sample surface, wherein the second light source is arranged toemit the pump beam toward a second sample surface, and wherein the firstsample surface is the same as the second sample surface.
 23. The systemaccording to claim 17, wherein the first light source is furtheroperable to: modulate the pump beam according to a first modulationfrequency.
 24. The system according to claim 23, wherein the firstmodulation frequency is greater than or equal to 100 kHz.
 25. The systemaccording to claim 17, wherein the first light source is a mid-infraredlaser having at least one emission wavelength within the range of 3 to25 micrometers.
 26. The system according to claim 17, wherein the secondlight source is a visible light laser having at least one emissionwavelength that is less than or equal to 800 nanometers.
 27. The systemaccording to claim 17, further comprising: a reflective objectiveadapted to focus the pump beam onto the region of the sample.
 28. Thesystem according to claim 17, further comprising: a refractive objectiveadapted to focus the probe beam onto the region of the sample.
 29. Thesystem according to claim 17, wherein the signal processing device is alock-in amplifier configured to: a signal from the detector; receive asignal indicative of a modulation frequency of the pump beam; determineat least one of (i) an amplitude and (ii) a phase of the probe beamcoming from the region of the sample; and generate the signal indicativeof the amount of infrared light absorbed by the region of the samplebased on the determined at least one of (i) the amplitude and (ii) thephase of the probe beam reflecting from the region of the sample. 30.The system according to claim 17, further comprising: a cell adapted forhigh infrared transmission and configured to hold the sample, whereinthe cell includes sapphire windows.
 31. A method for detecting infraredlight absorption in a sample with submicron resolution, the methodcomprising the steps: a) illuminating a region of the sample with a pumpbeam generated by a tunable source of mid-infrared radiation, wherein atleast a portion of the pump beam is absorbed by the region of the sampleat least one wavelength generated by the tunable source; b) illuminatingthe region of the sample with a probe beam generated by a second lightsource, wherein the probe beam has a wavelength shorter than the pumpbeam; c) collecting by a detector at least of a portion of the probebeam coming from the region of the sample; d) generating, based on probelight collected by the detector, a signal indicative of an amount ofinfrared light absorbed by the region of the sample with sub-micronspatial resolution; e) repeating steps (a)-(d) at a plurality ofwavelengths of the tunable source of mid-IR radiation.
 32. The methodaccording to claim 31, further comprising: generating from steps (a)-(e)a signal indicative of an absorption spectrum of the region of thesample; and based on the determined absorption spectrum, determining oneor more chemical compounds present in the sample.
 33. The methodaccording to claim 31, wherein the probe beam has a Gaussian beam widthof less than 2.33 micrometers.
 34. The method according to claim 31,wherein the probe beam has a Gaussian beam width of less than 0.7micrometers.
 35. A system for detecting infrared light absorption in asample with submicron resolution, the system comprising: a first lightsource operable to illuminate a region of the sample with pump beamgenerated by a tunable mid-infrared light source operable to generateradiation at a plurality of wavelengths; and wherein the sample absorbsat least a portion of the pump beam at at least one wavelength of thetunable light source; a second light source operable to illuminate theregion of the sample with a probe beam generated by a second lightsource, wherein the probe beam has a shorter wavelength than the mid-IRpump beam; a detector operable to collect at least a portion of theprobe beam having coming from the region of the sample; a signalprocessing device configured to generate a signal indicative of anamount of mid-infrared light absorbed by the region of the sample withsub-micron spatial resolution at a plurality of wavelengths of thetunable IR pump beam.
 36. The system according to claim 35, wherein thesystem is further configured to: generate a signal indicative of anabsorption spectrum of the sample based on the signal indicative of themid-IR absorption of the region of the sample at the plurality ofwavelengths of the tunable IR pump beam; and based on the determinedabsorption profile, determine one or more chemical compounds present inthe sample.
 37. The system according to claim 35, wherein the probe beamhas a Gaussian beam width of less than 2.33 micrometers.
 38. The systemaccording to claim 35, wherein the probe beam has a Gaussian beam widthof less than 0.7 micrometers.